Method for enhancing yield of recombinant protein production from plants

ABSTRACT

The present invention relates to a method for enhancing the yield of recombinant protein produced in genetically transformed plants. The invention most particularly relates to a method for preventing the undesirable proteolysis of recombinant proteins after harvest of the plant, during processing of the products from the plants. Especially, this invention focuses on introducing protease inhibitors in plants to prevent undesirable proteolysis of recombinant proteins at the time of cell disruption during the extraction process.

TECHNICAL FIELD

The present invention relates to a method for enhancing the yield ofrecombinant protein produced in genetically transformed plants. Theinvention most particularly relates to a method for preventing theundesirable proteolysis of recombinant proteins after harvest of theplant, during processing of the products from the plants. Especially,this invention focuses on introducing protease inhibitors in plants toprevent undesirable proteolysis of recombinant proteins at the time ofcell disruption during the extraction process.

BACKGROUND ART

Recombinant expression of proteins is widely used to produce proteins ofinterest. Commonly used host systems are bacteria, yeast, insect cells,mammalian cells, animals and plants. However, recombinant proteinexpression is often impaired due to a multitude of factors. Inparticular, the yield of recombinant protein production is closelyassociated with the stability of the protein during the accumulation andthe extraction processes.

In plants, several recombinant proteins have been produced with successbut the primary problem encountered is the low level of recombinantprotein recovery. One cause of low yield is the activity of proteasesthat degrade proteins.

In plants, interactions between recombinant proteins and proteases arenot well defined at this time, but it is known that plants possessseveral non-specific proteases in their vacuoles. Leaf vacuolarproteases that are active in the mildly acidic pH range, maysignificantly alter the stability and integrity of recombinant proteins,and then decrease the yield of production of intact proteins.

Plant proteases may degrade recombinant proteins during two criticalsteps of the process of protein production. The degradation mayoccur, 1) in planta, during accumulation of the protein, and 2) explanta, at the time of cell disruption during the extraction process.The latter may be of greater importance, since in this step, celldisruption liberates a pool of proteases from all parts and cellcompartments of the plant. For example, it has been reported that therice cystatin I (OC-1), a clinically useful protein, is accumulated in astable form in the cytoplasm of transgenic potato leaf cells, but isdegraded by proteases at the time of extraction (Michaud and Yelle,2000, Michaud Ed., Austin Tex., pp. 195-206).

The basic process for extracting recombinant proteins from plant leavesgenerally begins with disintegrating a plant biomass and pressing theresulting pulp to produce a green juice. The green juice typicallycontains various proteins including proteases and a green pigmentedmaterial. It is of no use to achieve a high accumulation of recombinantprotein in planta if the level ex planta, during the extraction processis decreased drastically by the activity of proteases. This inventionfocuses on the prevention of proteolysis occurring ex planta at the timeof cell disruption during the extraction process.

Various methods in the art are suggested to protect recombinant proteinagainst degradation by proteases.

So far, research has mainly focused on decreasing proteolyticdegradation in planta. For example, one strategy to overcome theproteolysis problem in plants is to target proteins to alternativeorganelles and direct their accumulation in sub-cellular compartmentswhere the protein is more stable. Different studies have demonstrated anincrease in intracellular accumulation of a protein of interest, such asantibodies or vicilin when targeted to the endoplasmic reticulum usingthe carboxy-terminal signal KDEL (Tabe et al., 1995, J. Plant Sci.73:2752-2759), instead of being targeted to the vacuole. However,although this strategy helps prevent proteolysis during expression ofrecombinant proteins, it does not reduce the risk of proteolysis at thetime of extraction. During extraction, plant cells are disrupted andthen release various compounds into the medium, including proteases,that may severely alter the integrity of recombinant proteins.

Another strategy is the alteration of proteolytic metabolism in planta.In one example, plants are genetically modified to decrease or eliminatethe activity of specific proteases such as vacuolar processing enzymes(VPE's) as disclosed in U.S. Patent application No. 2002/0108149. Inanother example, catabolic processes including proteolysis aresuppressed by delaying organ senescence (Int. Patent Publication No.WO01/61023). Again, these strategies may prevent degradation ofrecombinant proteins during their accumulation in planta, but do notreduce the risk of proteolysis during the extraction process.Additionally, these strategies are limited to alteration of proteolyticmetabolism or/and proteases that are non-essential for plantdevelopment.

Classical methods to reduce the degradation of recombinant proteins explanta during extraction, consist in quickly adjusting the pH of theextraction buffer (e.g. to pH 7) and/or in includinglow-molecular-weight protease inhibitors in the extraction buffer.

It is known in the art that an acidic pH increases the degradation ofprotein in the extraction mixture. The pH adjustment method is a viablemethod to limit the degradation of recombinant protein in the extractionmixture. However, this method is not effective with all recombinantproteins. Additionally, the use of an acidic pH to precipitate proteinsin the extraction mixture and to isolate the soluble fraction containingthe recombinant protein of interest, is a very useful method topartially purify these proteins, and thus maintaining pH to 7 is aconstraint one would wish to eliminate at industrial scale. In the caseof recombinant protein production in plants, this possibility is ofgreat interest since the vast diversity of proteins from plants and thestringent purity requirement in industrial and medical applicationsrequires an efficient and economical procedure for their isolation andpurification.

The other classical method to prevent proteolysis, which consists in theaddition of low-molecular-weight protease inhibitors, such asphenylmethyl sulfonyl fluoride (PMSF) or chymostatin in the extractionmixture, could be useful in a small-scale production.

However, this method is not economically suitable on an industrial scaleof plant recombinant protein production, where proteins need to beproduced cost-effectively in large amounts.

Considering the costly process of producing recombinant proteins inplants, it is desirable to obtain high production levels of recombinantproteins. Especially, since recombinant protein levels at the time ofcell disruption during the extraction process should be high, and it isof no use to achieve a high level of recombinant protein in planta ifthe resulting level after extraction is comparatively low. In thiscontext, new cost-effective methods are needed to reduce degradation ofrecombinant proteins by plant proteases released in the medium aftercell/tissue disruption.

DISCLOSURE OF THE INVENTION

One aim of the present invention is to provide a method for increasingthe recovery yield of a recombinant protein in plant cells withoutsignificantly altering the natural physiology of the plant cells,comprising neutralizing the activity or the action of at least one plantprotease involved in the degradation of the recombinant protein with aninhibitor released from the plant cell at the time said plant cells aredisrupted. The plant cells are from a plant or from an in vitro culture.It will be recognized by those skilled in the art that the neutralizingis partial or total, and can occur when processing the plant cells forextracting the recombinant protein, and that plant cells are disruptedwhen performing a process for extracting the recombinant protein. Theinhibitor is preferentially recombinantly produced in the plant cellstransformed with an expression cassette comprising a promoter operablylinked thereto. Also, the inhibitor can be linked to a leader peptide, asignal peptide or an anchorage peptide or a protein to lead or anchorsaid inhibitor to a cell part or extracellular compartment in a mannerto protect the recombinant protein from the activity of a plant proteaseduring the extraction process. For example, but not limited to, the cellpart can be an organelle selected from the group consisting of amitochondria, a chloroplast, a storage vacuole, the endoplasmicreticulum, and the cytosol. Also, the inhibitor can be encoded by a geneunder control of a constitutive or an inducible promoter or a tissue ordevelopment specific promoter.

Targeted proteases to be inhibited or neutralized can be selected fromthe group consisting of a cysteine protease, an aspartate protease, ametallo protease, a serine protease, a threonine protease, and amultispecific protease.

In accordance with one particular aspect of the present invention, theinhibitor significantly does not interfere with the activity of theprotease to preserve the physiology or the growth of the plant cells orplant containing the plant cells.

Another aspect of the invention is to provide a method for neutralizing,or modulating in planta an inhibitor is selected from the groupconsisting of an antibody or a fragment thereof, a sens-mRNA oranti-sens mRNA, an inhibitor of transcription or a regulator thereof, aninhibitor of translation or a regulator thereof, an inhibitor of leadingor signal peptide, an inhibitor of metabolic acquisition of activity ofa protease, a protease-specific protease, and an affinity peptideprotease leading to segregation to said protease into an organelle or acell compartment.

Preferentially, the genetically altered plant is an alfalfa or apotatoe.

The targeted proteases to be neutralized can be a chymostatin-sensitiveserine protease or a cystatin-sensitive cysteine protease.

Preferentially, the recombinant protein or inhibitor are produced innucleus or plastids of said plant cells.

Another aim of the present invention is to provide method for increasingthe recovery yield of a recombinant, protein in a plant comprising thesteps of:

-   -   a) allowing production of a recombinant protein in plant cells        genetically altered for modulating at least one genetic or        metabolic reaction to partially or totally neutralize action or        activity of at least one protease at the time of disrupting of        the plant cells; and    -   b) recovering the recombinant protein after disrupting of the        plant cells.

The plant cells are from a plant or from in vitro culture.

The action or activity of the protease can be neutralized by inhibitingits transcription or translation into an active protease, or by aninhibitor produced by the plant cells, or linking the recombinantprotein with a peptide or protein in manner to protect the recombinantprotein from the action or activity of the protease.

In accordance with the present invention there is provided a plant cellor a plant genetically altered to modulate at least one genetic ormetabolic reaction to partially or totally neutralize the action oractivity of at least one protease for improving the recovery of arecombinant protein from the plant cell or plant at the time the plantcell or cells of the plant are disrupted.

Another object of the present invention is to provide a method ofintroducing protease inhibitors in plants to prevent undesirableproteolysis of recombinant proteins at the time of cell disruptionoccurring or performed during the extraction process.

This invention is partially based on the identification of proteininhibitors efficient in inhibiting an important fraction of potato andalfalfa proteases found in crude extracts of leaves and stems. Targetprotease activities in potato and alfalfa have been tested forproteolytic on proteins of interest such as human fibronectin. Theseplant proteases show proteolytic activity against recombinant proteinsof interest and the present invention provides new strategies to alterthe undesirable activity of these proteases during the extractionprocess.

One object of the present invention is also to provide a method toenhance the yield of production of recombinant proteins in plants bypreventing proteolysis after cell disruption but without negativelyaltering the normal metabolism or development of the host plant.

Also one object of the present invention is to provide a method toprevent proteolysis of recombinant proteins at the time of celldisruption during the extraction process, this method allowing, forexample, the use of acidic pH in the extraction mixture to precipitateproteins and isolate a soluble fraction containing the recombinantprotein of interest.

Another goal of the invention is the judicious choice of the inhibitorto be expressed in the plant as well as its subcellular targeting, toinsure a sufficient accumulation of the inhibitor in planta and asatisfying stability of this inhibitor at the time of harvesting,stocking and extraction, in order to reach the optimal protection effectof recombinant proteins at the time of cell disruption during theextraction process.

According to one aspect of the present invention there is provided amethod for enhancing the yield of production of recombinant protein inplants or plant cells, this method comprising the step of obtainingplants or plant cells co-expressing at least (a) a recombinant protein,and (b) an inhibitor of endogenous plant proteases implicated in thedegradation of said recombinant protein, whereby the control expressionof the inhibitor specified at (b) enables the proteolytic degradation ofthe recombinant protein specified at (a) to be prevented or reducedthereby increasing the recovery yield of the recombinant protein,without altering negatively the metabolism or development of the plantor plant cells.

The inhibitor may be co-expressed in the plant with the protein ofinterest, or fused to the protein of interest. The inhibitor may beco-expressed with the recombinant protein in the same sub-cellularcompartment, or in a different one.

The use of antibodies or a fragment thereof as a protease-specificinhibitor is also another aspect of the present invention.

According to the method of the present invention, there is provided agenetic alteration, such as DNA fragment insertion into a plant toinhibit the expression of a protease. The genetic alteration may includelockout or silencing methods. The invention also includes methods inwhich the inhibitory effect is constitutive or inducible, which is madepossible by the use of constitutive or inducible promoters.

The present invention also provides a method in which a transgenic plantexpressing a recombinant protein of interest is harvested with atransgenic plant expressing at least one protease-specific inhibitor, inorder to protect the protein of interest against endogenous proteases ofthe plant released during the cell lysis and/or the extractionprocedure.

For the purpose of the present invention, the following terms aredefined below.

The term “recombinant protein” as used herein is intended to mean aprotein, peptide, or polypeptide that is produced by the plants or plantcells using recombinant techniques. The recombinant protein is producedthrough the expression of a corresponding transgene which has beenintroduced in the plants or plant cells to have genetically modifiedplants or plant cells and expressed therein.

Proteins or factors that can be recombinantly produced may for example,but not limited to, alpha.-, beta.- and .gamma.-interferons,immunoglobulins, lymphokines, such as interleukins 1, 2 and 3, growthfactors, including insulin-like growth factor, epidermal growth factor,platelet derived growth factor, transforming growth factor-.alpha.,-.beta., etc., growth hormone, insulin, collagen plasminogen activator,tissue plaminogen activator, thrombin, fibrinogen, aprotinin, bloodfactors, such as factors I to XII, histocompatibility antigens,collagen, gelatin, enzymes such as superoxide dismutase, or othermammalian proteins, particularly human proteins.

The terms “promoter” or “promoter region” or “transcriptional regulatorysequence” as used herein mean a DNA sequence, usually found upstream(5′) to a coding sequence, that controls expression of the codingsequence by controlling production of messenger RNA (mRNA) by providingthe recognition site for RNA polymerase and/or other factors necessaryfor initiation of transcription at the correct site. As contemplatedherein, a promoter or promoter region includes variations of promotersderived by means of ligation to various regulatory sequences, random orcontrolled mutagenesis, and addition or duplication of enhancersequences. The promoter region disclosed herein, and biologicallyfunctional equivalents thereof, are responsible for driving thetranscription of gene sequences under their control when introduced intoa host as part of a suitable recombinant vector, as demonstrated by itsability to produce mRNA.

The expressions “plant cell” or “plant part” as used herein is intendedto refer to plantlets, protoplasts, calli, roots, tubers, propagules,seeds, seedlings, pollen, any other plant tissues.

The term “protease” is intended to mean an enzyme that performs directlyor indirectly the degradation of polypeptides into smaller peptides,fragments or amino acids, or into a form leading to the loss of thestability or activity of a protein of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C illustrate the time-course degradation of NPTII (A),human fibronectin (B) and human haemoglobin (C) by a crude extract ofproteins of alfalfa leaves. The NPTII protein (A) was obtained throughstable expression and extraction from potato leaves. Commerciallyavailable fibronectin (B) and haemoglobin (C) were added to crudeextract of alfalfa leaves.

FIG. 2 illustrates the proteolytic activity of alfalfa (A) and potato(B) proteases in a gelatin-embedded polyacrylamide gel;

FIG. 3 illustrates the inhibition of specific alfalfa leaf proteases inalfalfa leaf with diagnostic and plant recombinant PIs;

FIG. 4 illustrates the inhibition of specific potato leaf proteases inpotato leaf with diagnostic and plant recombinant PIs;

FIGS. 5A and 5B illustrate the separation of alfalfa leaf proteases byion exchange chromatography (A), and the stabilization of humanfibronectin against a major protease fraction with chymostatin andα-1-antichymotrypsin (B);

FIGS. 6A, 6B and 6C illustrate the separation of a potato leaf cathepsinD-like activity by ion exchange chromatography (A and B), and itsinhibition by the aspartate proteinase inhibitor GST-CDI (C);

FIG. 7 illustrates the decrease in cathepsin D-like activity intransgenic potato lines expressing a tomato CDI transgene;

FIG. 8 illustrates the partial stabilization of recombinant NPTII in atransgenic potato line (CD21A) expressing a tomato CDI transgene, ascompared to a control plant; and

FIG. 9 illustrates variations of the strategy of recombinant proteaseinhibitor expression in plants to hinder protease activity after celldisruption, during the protein recovery process.

MODES OF CARRYING OUT THE INVENTION

The present invention provides new methods for enhancing the yield ofrecombinant protein recovered from transgenic plants or plant cells.

Also, the present invention is directed to a method for producing plantlines genetically altered to inhibit at least one protease forpreserving the integrity of a recombinant protein of interest at thetime of cell disruption during the extraction process.

Also, one object of the present invention is to provide a method forpreventing proteolysis of recombinant proteins at the time of celldisruption during the extraction process, this method allowing the useof acidic pH in the extraction mixture to precipitate proteins andisolate a soluble fraction containing the recombinant protein ofinterest.

In one embodiment of the invention a protease can be identified andtargeted to be inhibited as a protease specifically involved in thedegradation of a recombinant protein of interest during the extractionprocess.

In another preferred embodiment of the invention strategies tospecifically express and target the recombinant protein and the proteaseinhibitor are chosen so as to significantly not to affect or preservethe metabolism or development of the transgenic plant. It will beunderstood here that the normal physiology of a plant or plant cell inwhich conditions for inhibiting the activity or action of a protease atthe time of recovering, including cell lysis, the protein of interest,is preferentially not altered. For example, but not limited to, a plantin which genetic modification results in inhibition of a proteasetherein, will grow at the same rate than a non modified plant. Underanother aspect, the protein synthesis is also not altered by theconditions in the plant or plant cell resulting in the inhibition of aprotease when recovering or extracting a protein of interest.

In another embodiment of the invention, a protease inhibitor can betargeted to a subcellular compartment different from the naturallocalization of a targeted protease in order to preserve the vitalactivity of the protease during the growth of the plant, and promoteprotection of recombinant proteins at the time of cell disruption duringthe extraction process of the recombinant protein.

In accordance with the present invention, there is provided a methodthat will give conditions causing the inhibition, partial or total, ofthe action or the activity of the proteases at the time a protein ofinterest is recovered or extracted from a plant or a plant cell.Preferentially, the method makes use of protease inhibitors, and use ofsequences to genetically engineer plants or plant cells in a manner toprotect from the activity of a protease the recombinant proteinsproduced in these transgenic plants or plant cells. Another condition ofinhibiting the activity of a protease according to the present inventionis that the inhibitor binds directly the protein of interest to avoidthe protease to access the cleavage site for example, of binds directlythe protease in order to block its action or activity.

In another embodiment of the invention the inhibitor can be chosen fromthe group consisting of, but is not limited to, (i) inhibitors ofcysteine proteases, (ii) inhibitors of aspartate proteases, (iii)inhibitors of metallo proteases, (iv) inhibitors of serine proteases,(v) inhibitors of threonine proteases, and (vi) inhibitors with a broadrange of specificity, natural or hybrid.

Alternatively, the protease inhibition according to the invention, canbe performed in changing the specificity of the protease itself or thecondition that cause changes in the specificity of the protease for theprotein of interest during its recovering or extraction. The specificitychanging or the protease for the protein of interest will preferentiallynot affect its activity naturally occurring in a plant or plant cell.

Different strategies can be employed to engineer plants. For example,this can be carried out, without limiting it thereto, by 1) inserting aprotease inhibitor encoding gene into the genome of a plant, Anotherembodiment of the present invention is to provide a method in which anygene encoding a potent protease inhibitor may be introduced into thegenome of a plant to reduce proteolytic activity during the extractionprocess which is desirable for the high-yield production of recombinantproteins. Examples of protease inhibitors that could be introduced intoplants consist of, but are not limited to, the plant cystatins OCI, OCIIand TMC-8, the human serpin alpha-1-anti-chymotrypsin (AACT), and theaspartate type inhibitor CDI (Tomato cathepsin-D inhibitor). Forexample, human serpin alpha-1-anti-chymotrypsin (AACT) could be used toinhibit alfalfa endogenous proteases while tomato CDI could be expressedin potatoe to block the endogenous aspartae proteinase. A method forintroducing a protease inhibitor in alfalfa and potato is exemplifiedhereinbelow.

The inhibitor can be alternatively a protease propeptide.

One way to achieve protease inhibition, is also the production, intransgenic plant, of a specific antibody or an antibody fragmentdirected to a protease that will hinder its normal activity. This methodof inhibition is dependent on the capacity of the antibody to bind toits antigen in the plant cell. Hence, it is required that the plantproduces the antibody, which can be achieved by genetically transformingthe plant with the transgene or transgenes needed to produce an activeimmunoglobulin. The production of antibodies or fragments thereof inplants is known of those skilled in the art since different antibodieshave been expressed in transgenic plants including immunoglobulins (IgG,IgA and IgM), single chain antibody fragment (ScFv), fragment antigenbinding (Fab), and heavy chain variable domains.

The antibody or a fragment thereof could be targeted to a differentsubcellular compartment from the natural localization of the targetedprotease in order to preserve the vital activity of the protease duringgrowth of the plant, and to promote protection of the recombinantprotein specifically at the time of extraction or cell lysis.

One embodiment of the present invention is to provide a method thatutilizes at least one DNA fragment to inhibit the expression of anendogenous protease in a genetically altered plant producing arecombinant protein.

According to another aspect of the invention, plants or plant cells areobtained with a vector useful for plant or plant cell transformation,comprising a DNA sequence encoding the recombinant protein and a DNAsequence encoding the inhibitor.

According to one aspect of the invention, transgenic plants or plantcells are obtained by transformation of whole plant, plant cells, plantprotoplasts or plant plastids with one or more useful vectors comprisingat least: (a) a first DNA fragment harbouring a DNA sequence encoding arecombinant protein of interest operably linked to a first promoter,fused or not to a targeting peptide to direct the protein to aparticular subcellular or extracellular compartment of the plant orplant cells; and (b) a second DNA fragment harbouring a DNA sequenceencoding a protease inhibitor operably linked to a second promoter,fused or not to a targeting peptide to direct the inhibitor to aparticular subcellular or extracellular compartment of the plant orplant cells.

According to another aspect of the invention, plants or plant cells areobtained by crossing a first plant comprising (a) a first DNA fragmentharboring a DNA sequence encoding a recombinant protein operativelylinked to a first promoter, fused or not to a first targeting peptide todirect the protein to a particular subcellular or extracellularcompartment of the plant or plant cells, with a second plant containing(b) a second DNA fragment harbouring a DNA sequence encoding a proteaseinhibitor operably linked to a second promoter, fused or not to a secondtargeting peptide to direct the inhibitor to a particular subcellular orextracellular compartment of the plant or plant cells.

In one embodiment of the invention, the presence or absence of a signalpeptide achieves targeting of the protease inhibitor to the samesubcellular or extracellular compartment as the recombinant protein ofinterest. Alternatively, the presence or absence of a signal peptideenables to target the inhibitor to a subcellular or extracellularcompartment that is different from the recombinant protein of interest.

In one embodiment of the invention, targeted sub-cellular orextracellular compartments of the plant are chosen from the group of,but not limited to, mitochondria, plastids, storage vacuoles,endoplasmic reticulum, cytosol, and extracellular compartment.

Also, according to another aspect of the invention, transgenic plants orplant cells are obtained by genetic transformation of a plant or plantcell with a vector suitable for plastid transformation comprising theDNA sequence encoding the recombinant protein and the DNA sequenceencoding the inhibitor operably linked to a promoter operative in theplastid.

Also, in one embodiment of the invention, the protease inhibitorencoding gene may be co-inserted in the plant genome with the gene ofthe protein of interest, in the same sub-cellular compartment or not.The inhibitor may be fused to the recombinant protein to be produced inthe plant. A plant expressing one or several protease inhibitors may becrossed with a plant expressing the recombinant protein.

In another aspect of the invention, transgenic plants or plant cells areobtained by genetic transformation with a vector comprising a DNAsequence encoding the recombinant protein fused to a DNA sequenceencoding the protease inhibitor operably linked with a unique promoter,and which optionally comprises the fusion of a targeting peptide todirect the fused protein and inhibitor to a particular subcellular orextracellular compartment of the plant or plant cells.

In another embodiment of the invention, expression vectors used toperform the method according to the invention may include a promoterthat can be constitutive, inducible, development specific, tissuespecific, or stress specific.

Also, in order to perform the method according to the invention, theactivity or expression of a protease can be directly or indirectlygenetically altered.

Also, part of the invention is the use of constitutive but alsoinducible promoters to control the expression of the inhibitor. Forexample, the inhibitor could be induced, or its synthesis, at the timeof harvesting only, by the addition of the inducing agent priorharvesting.

Alternatively, according to another aspect of the invention the methodmay involved the exogenous induction of an endogenous plant inhibitor toinhibit a specific protease inhibitor at the time of harvesting toincrease the recovery yield of the recombinant protein.

In accordance with the present invention, are provided methods forproducing plant lines for molecular farming. Any plant species can beused to perform any method, strategy, or approach described herein topartially or totally inhibit the action of a protease against arecombinant protein of interest.

Of particular interest, the present invention can be applied to alfalfaor potato.

EXAMPLES

The present invention will be more readily understood by referring tothe following examples, that are given to illustrate the inventionrather than to limit its scope.

Example I Degradation of NPTII Protein by Plant Leaf Proteases

Materials and Methods

The hypothesis that degradation of specific recombinant protein can bedecreased by the expression of a exogenous protease inhibitor was testedusing a simple model. The neomycin phosphotransferase (NPTII) proteinwhich is often use as selectable marker of transgenic plants wasexpressed in potato without the presence of any protease inhibitorprotein and the degradation of the NPTII protein was monitored. In orderto mimic the situation where a protease inhibitor gene would be presentand expressed on the same construct as the nptII gene, a proteaseinhibitor gene, the tomato cathepsin-D inhibitor CDI (Werner et al,1993, Plant Physioly 103:1473), was introduced beside the NPTII gene butwithout any promoter hence prohibiting CDI gene expression.

The tomato CDI-encoding DNA sequence was isolated from the expressionvector pGEX-3X/CDI (Brunelle et al. 1999, Arch. Insect Biochem Physiol.42:88-98) by digestion with BamHI and EcoRI, and subcloned between theBamHI and EcoRI cloning sites of the commercial vector pCambia 2300(CAMBIA, Canberra, Australia). Axenically-grown plantlets of potato(Solamum tuberosum L. cultivar Kennebec) were used as source materialfor genetic transformation. The plantlets were maintained on MSmultiplication medium (Murashige and Skoog 1962, Physiologia Plantarum15:473-497) supplemented with 0.8% (w/v) agar (Difco, Detroit, Mich.)and 3% (w/v) sucrose, in a tissue culture room at 22° C. under a lightintensity of 60 μmol/m²/s and a 16 h/day photoperiod provided by coolfluorescent lights. Leaf discs of about 10 mm in diameter weregenetically-transformed using the bacterial vector Agrobacteriumtumefaciens LBA4404 as described by Wenzler et al. (1989, Plant Sci.63:79-85), except that cefotaxime, instead of carbenicillin, was usedfor A. tumefaciens growth control. Regenerated shoots were transferredonto selection medium with kanamycin and cefotaxime, for rootregeneration and plantlet multiplication. For acclimation, the plantletswere transferred for 14 days in a growth chamber under a 24°/21° C.day/night temperature cycle, a 12-h L:D photoperiod, a light intensityof 200 mmol/m²/s and a relative humidity of 60%, before beingtransferred in greenhouse under standard growth conditions. Integrationof the nptII (marker) transgene in kanamycin-resistant plants wasconfirmed by PCR, using DNA extracted from the fourth, fifth and sixthleaves (from the apex) of ˜30-cm potato plants, according to Edwards etal. (1991, Nuc. Acids Res. 19:1349).

Protein extracts were prepared from PCR positive plants and subjected toa time course experiment where the degradation of the NPTII protein wasmonitored by Western analysis using a commercially available antibody.FIG. 1A illustrates the degradation of NPTII protein by potato leafproteases in crude extracts from control transgenic lines expressing thenptII gene and containing the CDI gene without promoter. Detection ofNPTII protein was performed by Wester blotting techniques. As seen onthe Western blot (FIG. 1A), NPTII protein degradation is observed withinthe first 10 min. of incubation.

Example II Degradation of Clinically-Useful Proteins by Plant LeafProteases

Materials and Methods

Other recombinant proteins may be targeted for degradation byproteolysis during the extraction procedure. In particular, thisdegradation may have a very negative effect for the recovery of plantmade pharmaceuticals. To illustrate that this process which occurs inpotato can also be found in other plants, the degradation of clinicallyuseful proteins was monitored in leaf extracts of alfalfa. Thisexperiment involved the addition of commercially available proteins toalfalfa leaf extract in vitro and the monitoring of the degradation ofthese proteins by Western analysis over a time period. In a firstexperiment (FIG. 1B), in vitro degradation of human fibronectin in thepresence of alfalfa proteases was monitored by mixing 5 μl of alfalfa(cultivar Saranac) leaf extract prepared in 50 mM Tris-HCl pH 7.0 (1:3w/v) containing 10 mM β-mercaptoetlianol, with 2 μg of fibronectin(Boehringer Mannheim, cat # 1080938). The mixture was incubated at 37°C. and the reaction was stopped by adding 5 μl of SDS-PAGEdenaturing/loading buffer. The protein samples (T=0 and T=1 hr) wereloaded on a 10% (w/v) SDS-PAGE gel, and electro-transferred onto anitrocellulose membrane. The substrate proteins and their proteolyticfragments were immunodetected with polyclonal antibodies against humanfibronectin (Sigma Aldrich, cat # F3648).

In a second experiment (FIG. 1C), in vitro stability of humanhaemoglobin in the presence of alfalfa proteases was monitored by mixing20 μg of alfalfa leaf extract prepared with 20 mM Mops, pH 7,5,containing 0,1% Triton X-100, 2 mM PMSF and 10 μM chymostatin, with 200ng of haemoglobin (Sigma, cat # H-7379), for a final volume of 8 μl. Themixture was incubated at room temperature and the reaction was stoppedby adding 2 μl of denaturing/loading buffer 5× with β-mercaptoethanol atdifferent times (0′, 15′, 30′, 1 h and 1 h30). The protein samples wereloaded on a 15% (w/v) SDS-PAGE gel and electro-transferred onto a PVDFmembrane. The substrate protein was immunodetected with monoclonalantibodies against human hemoglobin (Fitzgerald Cat # 10H03). The lane“Std” on the gel corresponds to 200 ng pure hemoglobin. In summary, FIG.1 illustrates the degradation of fibronectin (B) and hemoglobin (C) inthe presence of alfalfa leaf extracts, showing the hydrolytic effect ofplant's endogenous proteases against these proteins. Fibronectin, forinstance, is readily degraded by alfalfa (cultivar Saranac) endogenousproteases to lead intermediates finally hydrolyzed (FIG. 1B). Hemoglobinis also degraded after a 30 min. incubation with alfalfa proteases (FIG.1C).

Example III Identification of Major Protease Activities in Plant LeafExtracts

Materials and Methods

There are several kind of proteases found in different plant species. Inorder to characterize the major protease activities found in alfalfa andpotato, crude protein extracts were obtained from the leaves of thesetwo species. FIG. 2 illustrates the hydrolytic action of endogenousalfalfa (A) and potato (B) leaf proteases (arrows) on the degradation ofgelatin. Soluble proteins were extracted (1:3 W/V) from alfalfa(cultivar Saranak) or potato (cultivar cultivarKennebec) leaves with 50nM Tris-HCl pH 7.5, and resolved under non-reducing conditions on a 10%(w/v) SDS-polyacrylamide slab gel embedded with 0.1% (w/v) gelatin(Michaud et al., 1993, Electrophoresis 14:94-98). Proteinaserenaturation was carried out by incubating the gels for 30 min at 25° C.in 2.5% (v/v) Triton X-100. Gelatinase reaction was activated by placingthe gels in 100 nM citrate phosphate pH 6.0, containing 0.1% TritonX-100 and 5 mM L-cysteine, for 30 min at 37° C. Proteinases werevisualized as clear (lysis) bands against a blue background, afterstaining with Coomassie Brilliant Blue.

This detection method would easily enable the identification of aspecific protease inhibitor activity towards one of more proteaseactivities obtained. One skilled in the art could perform similarprotein extract, add the specific protease inhibitor, and detect on thegelatin gel the disappearance of lysis band which would indicate thatthe protease inhibitor used was able to inactivate this specificprotease activity.

Example IV Effect of Various Protein Inhibitors on Specific PlantProtease Activities

Materials and Methods

Using the method described in example III, it may be possible toidentify which protease activity is responsible for the degradation of aspecific clinically useful protein. From there, it would be interestingto be able to find a specific protease inhibitor to selectively abolishthe protease activity. The use of synthetic fluorometric proteasesubstrates was investigated towards this application. Fluorimetricprotease substrates are useful to determined the potential of variousdiagnostic or recombinant PIs on the inhibition of specific plantproteases. Leaf proteins were extracted (1:3 w/v) in 50 mM Tris-HCl pH7.5 containing 10 mM 3-mercaptoethanol, and protein content was adjustedto a final concentration of 1 mg/ml with extraction buffer. A masterreaction mix was prepared by mixing 1080 μl extraction buffer, 108 μlplant extract and 12 μl of either 1 mM Ala-Ala-Phe-MCA, 1 mMsuc-Ala-Ala-Pro-Phe-MCA, 1 nM suc-Leu-Val-Tyr-MCA or 1 mM Bz-Arg-MCA.One hundred μl of the master mix were dispensed in 96-well microplatesand 5 μl of 100 mM PMSF (inhibitor of serine proteases), 1 mM aprotinin(inhibitor of serine proteases), 10 mM chymostatin (inhibitor of serineproteases and some cysteine proteases), 1 mg/ml α-1 antichymotrypsin(inhibitor of chyinotrypsin-like proteases), 10 mM leupeptin (inhibitorof trypsin-like proteases and some cysteine proteases), 1 mM pepstatin(inhibitor of aspartate proteases), 100 mM E-64 (inhibitor of cysteineproteases), recombinant CDI (cathepsin-D inhibitor; inhibitor ofaspartate proteases), recombinant OCI (oryzacystatin I; inhibitor ofcysteine proteases), recombinant CCII (corn cystatin 2; inhibitor ofcysteine proteases) and recombinant PMC8 (potato multicystatin domain 8;inhibitor of cysteine proteases) were finally added to the reactionmixture. Fluorescence intensity was measured 100 times over a 5,000-secperiod at 30° C. using a Fluostar Polastar Galaxy™ fluorimeter (BMG LabTechnologies), with excitation and emission filters of 485 nm and 520mm, respectively. Protease activity, expressed in units of fluorescenceper min., corresponded to the slope of the emission curve. As shown inFIGS. 3 and 4, various types of proteases may be considered as possibletargets to decrease protease activities from alfalfa and potato leaves,including serine (e.g., PMSF-, aprotinin, chymotrypsin- andchymostatin-sensitive), cysteine (E-64/cystatin-sensitive) and aspartate(pepstatin-sensitive) proteases.

Example V Inhibition of Fibronectin Proteolysis in Alfalfa Leaf Extracts

Materials and Methods

The human fibronectin was shown to be susceptible to proteasedegradation in alfalfa leaf extract (FIG. 1C). The following step was todemonstrate the use of various protease inhibitors to inhibit thefibronectin degradation. The stability of fibronectin was significantlyincreased by inhibiting alfalfa proteases with the serine-type inhibitorα-1 antichymotrypsin (FIG. 5). Firstly, a protein extract from alfalfaleaves was separated by chromatography to isolate a specific fractioncontaining the greatest protease activity. Alfalfa (cultivar Saranac)leaves were extracted by grinding in liquid nitrogen andresolubilization in 50 mM Tris-HCl, pH 6,8, containing 10 mMβ-mercaptoethanol. The crude extract was centrifuged for 15 min at 10000g at 4° C., and the supernatant was filtered through a 0.3 μm pore sizefilter. Fifteen mg of leaf proteins were then loaded on of a Mono-Q FPLCcolumn (Pharmacia) equilibrated with extraction buffer. Proteins wereeluted with a linear gradient of KCl (0 to 0.7 M) in extraction buffer,at a flow rate of 2 ml/min. Fractions of 500 μl were collected, andsample of each fraction was loaded onto a gelatin/PAGE gel (FIG. 5A).Fraction #8, which caused the highest proteolysis of gelatin in gel, wasuse to assess the protective effect of α-1 antichyinotrypsin.

Secondly, the identified fraction #8 was used in conjunction withvarious protease inhibitors to identify potential candidates for theinhibition of fibronectin proteolysis. In the experiment illustrated inFIG. 5B, 5 μl of fraction #8 was mixed with 350 ng of fibronectin andincubated at 37° C. for 15 min, in the presence of 2 μl H₂O (lane 2), 2μl of 10 mM chymostatin (lane 3) or 2 μl α-1 antichymotrypsin (lane 4).The control (lane 1) contained 5 μl extraction of buffer instead ofalfalfa proteases. The reaction was stopped after 15 min. andfibronectin was immunodetected as in FIG. 1C. As shown in FIG. 5B, thefraction eluted by Mono-Q chromatography caused significant proteolysisof fibronectin, but was prevented by inhibitors of serine-likeproteases, chymostatin and α-1 antichymotrypsin. Note that both protein(α-1 antichymotrypsin) and chemical (chymostatin) molecules wereefficient to decrease degradation of fibronectin.

Example VI Inhibition of Cathepsin D-Like Protease Activity by aSpecific Aspartate-Type Protease Inhibitor in Potato Leaf Extract

Materials and Methods

Similarly to Example V, soluble proteins were prepared from potato(cultivar Kennebec) leaves, separated by Mono-Q chromatography, andsubmitted to gelatin/PAGE (FIG. 6A), as described in FIG. 5A. Proteaseactivity was determined for each chromatographic fraction by fluorimetryusing a cathepsin D-specific substrate(MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2) at afinal concentration of 6 μM (FIG. 6B). As depicted in FIG. 6C, proteaseactivity in the potato leaf protein fraction showing the highestcathepsin D-like activity (fraction # 13) was dramatically altered bythe aspartate-type inhibitor tomato cathepsin D inhibitor ‘CDI’,identifying CDI-sensitive proteases as interesting targets for thedevelopment of strategies aimed at protecting protein integrity via theinhibition of the plant's endogenous proteases. Noteworthy, our dataalso show that the inhibition of a single protease (or protease group)may be sufficient to protect a significant part of the proteins presentin crude extracts, despite the presence of other (insensitive) proteasesin the medium.

Example VII Stabilization of Recombinant Proteins by the EctopicExpression of a Tomato Cathepsin D Inhibitor in Potato

Materials and Methods

To assess the impact of ectopically expressing a recombinant proteaseinhibitor in the plant on the activity of endogenous proteases duringextraction (ex vitro), a cathepsin D inhibitor from tomato, tomato CDI(Werner et al. 1993, Plant Physiology 103:1473), was integrated into anexpression vector and stably expressed into potato (cultivar Kennebec).Transgenic controls expressing the selection marker neomycinephosphotransferase (NPTII) but no CDI were also devised by integratingthe CDI transgene with no promoter. The tomato CDI-encoding DNA sequencewas isolated from the expression vector pGEX-3X/CDI (Brunelle et al.1999, Arch. Insect Biochem. Physiol. 42:88-98) by digestion with BamHIand EcoRI, and subcloned between the BamHI and EcoRI cloning sites ofthe commercial vector pCambia 2300 (CAMBIA, Canberra, Australia). TheCaMV 35S promoter was isolated from the commercial plasmid pBI-121(Clontech, Palo Alto, Calif.) using a BamHI/SalI treatment, and thenligated between the BamHI and SalI cloning sites of the pCambiaconstruct including the CDI transgene. Transgenic controls (SPCD lines)expressing the selection marker neomycine phosphotransferase (NPTII) butno CDI were devised by integrating the CDI transgene with no promoter.Transformation of potato plants were performed as indicated in ExampleI. Expression of the CDI transgene in transgenic lines was monitored byRT-PCR and Northern blotting, using total RNA extracted from the fourth,fifth and sixth leaves of nptII transgene-positive plants, as describedby Logemann et al. (1987, Anal Biochem. 163:16-20).

The cathepsin D-like activity was determined in transgenic potato plantexpressing low (Kennebec, SPCD4 and SPCD7) or high (CD3A, CD18A, CD21A)levels of CDI mRNA. Leaf proteins were extracted as in Example IV.Fluorimetric assays of cathepsin-D activity were performed as in ExampleVI. As shown in FIG. 7, cathepsin D-like activity was significantlylowered in transgenic potato line expressing the CDI transgene. As shownby Western blotting with an appropriate polyclonal antibody (FIG. 8),degradation of the recombinant marker protein NPTII by potato leafproteases in crude protein extracts from transgenic lines expressinghigh levels of recombinant CDI mRNA (clone 21A) was significantlydecreased, compared to the degradation pattern observed for thetransgenic control line, SPCD4. While NPTII protein can still bedetected in the CD21 transgenic plant extract expressing the CDI at highlevel after 50 min, it is totally degraded only 10 min after incubationin the control line containing the promoter less CDI construct (SPCD).From a practical viewpoint, this observation shows that aspartateproteinase activity in potato leaf extracts is effectively inhibited bytomato CDI, protecting the recombinant protein from hydrolysis by thisenzyme.

As described above for alfalfa and potato, plant leaf cells contain aconsiderable amount of non-specific proteases released in the mediumduring extraction. It is generally assumed that most of non-specificproteolytic activities in plant leaf cells are accounted for byproteases active in the acidic-to-mildly acidic pH range, usuallybelonging to the cysteine and aspartate class of proteolytic enzymes. Itappears from the data presented here that different types ofproteases—for instance CDI and chymostatin-sensitive proteases—may havea significant impact on the stability of useful proteins. As mostnon-specific proteases are often found in cell compartments other thanthe cytoplasm, inhibitors active against these proteases (e.g. tomatoCDI or α1-antichymotrypsin) may be expressed in the cytoplasmiccompartment of leaf cells (or elsewhere) in such a way that they do notnegatively interfere with the host plant's metabolism in vivo, thenready to act against endogenous proteases after cell breakage during therecovery process.

In practice, two different strategies may be used to achieve this goal(FIGS. 9B and 9C). A first strategy consists in developing transgeniclines of alfalfa expressing an appropriate protease inhibitor, and thenusing this line as an “anti-proteolysis” (or “low-proteolysis”) factoryfor the generation of double transformants expressing useful proteins(FIG. 9B). A second strategy consists in designing fusion proteinscomprising the candidate protease inhibitor and the protein of interest,linked by a protease-sensitive cleavage site allowing cleavage of thefusion and recovery of the free proteins (FIG. 9C). For Strategy 1, theprotease inhibitor-expressing transgenic line then serves as a‘universal’ factory for the production of heterologous proteins inalfalfa. Strategy 2 is more specific, as gene fusions are devised foreach particular protein to express, but a single transformation step issufficient to protect the protein. In both cases the companion inhibitoris present in the plant's cells in vivo, then ready to inhibit anyactive plant target protease after disruption of cell compartmentsduring extraction.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. A method for increasing the recovery yield of a recombinant proteinin plant cells without significantly altering the natural physiology ofsaid plant cells, comprising neutralizing the activity or the action ofat least one plant protease involved in the degradation of saidrecombinant protein with an inhibitor released from said plant cell atthe time said plant cells are disrupted.
 2. The method of claim 1,wherein said plant cells are from a plant or from an in vitro culture.3. The method of claim 1 wherein said neutralizing is partial or total.4. The method of claim 1 wherein said neutralizing occurs whenprocessing said plant cells for extracting said recombinant protein. 5.The method of claim 1, wherein said plant cells are disrupted whenperforming a process for extracting said recombinant protein.
 6. Themethod of claim 1, wherein said protease is selected from the groupconsisting of a cysteine protease, an aspartate, protease, a metalloprotease, a serine protease, a threonine protease, and a multispecificprotease.
 7. The method of claim 1, wherein said inhibitor isrecombinantly produced in said plant cells transformed with anexpression cassette comprising a promoter operably linked thereto. 8.The method of claim 1, wherein said inhibitor is linked to a leaderpeptide, a signal peptide or an anchorage peptide or a protein to leador anchor said inhibitor to a cell part or extracellular compartment ina manner to protect said recombinant protein from the activity of aplant protease during the extraction process.
 9. The method of claim 7,wherein said inhibitor does not interfere with the activity of saidprotease to preserve the physiology or the growth of said plant cells orplant containing said plant cells.
 10. The method of claim 7, whereinsaid cell part is an organelle selected from the group consisting of amitochondria, a chloroplast, a storage vacuole, the endoplasmicreticulum, and the cytosol.
 11. The method of claim 7, wherein saidinhibitor is selected from the group consisting of an, antibody or afragment thereof, a sens-mRNA or anti-sens mRNA, an inhibitor oftranscription or a regulator thereof, an inhibitor of translation or aregulator thereof, an inhibitor of leading or signal peptide, aninhibitor of metabolic acquisition of activity of a protease, aprotease-specific protease, and an affinity peptide protease leading tosegregation to said protease into an organelle or a cell compartment.12. The method of claim 8, wherein said genetically altered plant is analfalfa or a potatoe.
 13. The method of claim 1, wherein said proteaseis chymostatin-sensitive serine protease.
 14. The method of claim 1,wherein said protease is a cystatin-sensitive cysteine protease.
 15. Themethod of claim 1, wherein said inhibitor is a protease inhibitor. 16.The method of claim 1, wherein said plant cells are genetically altered17. The method of claim 1, wherein said neutralizing is performed by aninhibitor encoded by a gene under control of a constitutive or aninducible promoter or a tissue or development specific promoter.
 18. Themethod of claim 3 or claim 5, wherein said recombinant protein orinhibitor are produced in nucleus or plastids of said plant cells.
 19. Amethod for increasing the recovery yield of a recombinant protein in aplant comprising the steps of: a) allowing production of a recombinantprotein in plant cells genetically altered for modulating at least onegenetic or metabolic reaction to partially or totally neutralize actionor activity of at least one protease at the time of disrupting of saidplant cells; and b) recovering said recombinant protein after disruptingof said plant cells.
 20. The method of claim 19, wherein said plantcells are from a plant or from in vitro culture.
 21. The method of claim19, wherein said action or activity of said protease is neutralized byinhibiting its transcription or translation into an active protease, orby an inhibitor produced by said plant cells, or linking saidrecombinant protein with a peptide or protein in manner to protect saidrecombinant protein from the action or activity of said protease.
 22. Aplant cell or a plant genetically altered to modulate at least onegenetic or metabolic reaction to partially or totally neutralize theaction or activity of at least one protease for improving the recoveryof a recombinant protein from said plant cell or plant at the time saidplant cell or cells of said plant are disrupted.
 23. The plant cell orplant of claim 22, wherein said modulation inhibits the transcription ortranslation of a gene encoding for a protease, or neutralizes a proteasewith a protease inhibitor produced in said plant or plant cell.
 24. Theplant cell or plant of claim 22, wherein said recombinant protein orprotease inhibitor is linked to a leader peptide, a signal peptide orprotein in manner to improve protection of said recombinant protein fromat least one protease.